Many bacterial pathogens of the intestinal tract have as an early and necessary step in initiation of disease the ability to penetrate gut epithelial cells. My lab's work in the early 1980's established that Shigella~s genetic machinery to trigger invasion is encoded on a large virulence-associated plasmid. More recent studies have shown that the invasion ability of other enteric bacteria is chromosomally encoded. This project is aimed at understanding the prokaryotic and eukaryotic requirements for bacterial internalization, with the ultimate aim being a thorough molecular definition of the events involved in bacterial invasion of eukaryotic tissues. Current experimental approaches involve the use of assays measuring bacterial entry into cultured lines of human epithelial cells of various tissue origins. Biochemical inhibitors of prokaryotic structure/function or of eukaryotic cell processes are employed in these tissue culture invasion assays to examine the requirements for bacterial uptake. Direct visualization of bacterial entry is measured via transmission electron microscopy, video microscopy, fluorescent microscopy, and confocal microscopy. In addition to the mechanistic approaches described above, genetic techniques are employed to clone the responsible bacterial genes. Eukaryotic receptors for bacterial ligands and specific eukaryotic cell responses to bacterial invasion are measured via inhibitor competition assays, ligand binding assays, and mRNA analyses of infected eukaryotic cells. The information gained from each of these approaches is integrated to provide a mechanistic understanding of bacterial entry for each pathway studied.